Project Summary Astrocytes are evolutionarily conserved and constitute a substantial proportion of the cells in the brain, yet our understanding of their identities and functions is far less comprehensive than for neurons. Astrocytes are thought to be important for regulating the formation and function of neuronal circuits, playing multiple roles in neurotransmitter reuptake, ionic buffering, metabolic support, and plasticity. Evidence from both mammals and Drosophila has also indicated that astrocytes are diverse in both function and morphology. However, the underlying genetic mechanisms and the functional importance of this diversity remain unclear. The focus of this grant is to characterize the functional and molecular diversity of astrocytes in the adult Drosophila central brain and their role in sleep, a conserved innate behavior. In Aim 1, I will systematically investigate genes regulating astrocyte heterogeneity, specifically their regional specification and morphology. I will also use single cell RNA sequencing to identify differentially expressed genes within astrocytes and visualize the spatiotemporal pattern of gene expression in astrocytes in the central brain. Completion of this aim will lay the foundation for a comprehensive cataloging of astrocyte identity and morphology in Drosophila. In Aim 2, I will study the role of local astrocyte-neuron interactions in regulating sleep and arousal using in vivo calcium imaging, electrophysiology and behavioral assays. In Aim 3 during the R00 phase, I will generate tools to make stable intersectional reagents for targeting of specific astrocyte populations that will be broadly useful for labeling and functional manipulation. I will use these tools to further dissect the diverse roles of astrocytes in regulating sleep behavior. The research arising from this proposal will greatly expand our knowledge of astrocyte biology, by 1) identifying the genes and molecules responsible for astrocyte function and diversity, 2) elucidating their interactions with neuronal circuits and how they regulate behavior, and 3) development tools to target and manipulate this important cell type. Importantly, the training plan, including coursework, professional development, and mentorship from experts in neurobiology, astrocytes, and genetic tool-building, will prepare me for a future career as an independent academic neuroscientist.